xcex944-pregneine-11xcex2,17xcex1,21-triol-3,20-dione (hydrocortisone) is an important pharmaceutical steroid, used for its pharmacological properties as a corticosteroid and as a starting compound for the preparation of numerous useful steroids, particularly other corticosteriods. Hydrocortisone is produced in the adrenal cortex of vertebrates and was originally obtained, in small amounts only, by a laborious extraction from adrenal cortex tissue. Only after structure elucidation were new production routes developed, characterized by a combination of chemical synthesis steps and microbiological conversions. Only because the starting compounds which are employed such as sterols, bile acids and sapogenins are abundant and cheap, the present processes afford a less expensive product, but these still are rather complicated. Several possibilities were envisaged to improve the present processes, and also biochemical approaches have been tried.
One attempt was to have a suitable starting steroid converted in an in vitro biochemical system using the isolated adrenal cortex proteins which are known to be responsible for the enzymatical conversion in vivo of steroids to hydrocortisone. However, the difficult isolation of the proteins and the high price of the necessary cofactors, appeared to be prohibitive for an economically attractive large scale process.
Another approach was to keep the catalyzing proteins in their natural environment and to have the adrenal cortex cells produce the desired hydrocortisone in a cell culture. But due to the low productivity of the cells, in practice, it appeared to be impossible to make such a biochemical process economically attractive.
The in vivo process in the adrenal cortex of mammals and other vertebrates constitutes a biochemical pathway, which starts with cholesterol and via various intermediate compounds eventually affords hydrocortisone (see FIG. 1). Eight proteins are directly involved in this pathway, five of them being enzymes, among which four cytochrome P450 enzymes, and the other three being electron transferring proteins.
The first step is the conversion of cholesterol to 3xcex2-hydroxy-5-pregnene-20-one (pregnenolone). In this conversion, a mono-oxygenase reaction, three proteins are involved: side-chain cleaving enzyme (P450SCC, a heme-Fe-containing protein), adrenodoxin (ADX, a Fe2S2 containing protein) and adrenodoxin reductase (ADR, a FAD-containing protein).
Besides cholesterol as a substrate, the reaction further requires molecular oxygen and NADPH. Subsequently, pregnenolone is converted by dehydrogenation/isomerization to xcex944-pregnene-3,20-dione (progesterone). This reaction, catalyzed by the protein 3xcex2-hydroxy steroid dehydrogenase/isomerase (3xcex2-HSD), requires pregnenolone, and NAD+.
To obtain hydrocortisone, progesterone subsequently is hydroxlated at three positions which conversions are catalyzed by mono-oxygenases. In the conversions of progesterone into 17xcex1-hydroxy progesterone, two proteins are involved:
steroid 17xcex1-hydroxylase (P45017xcex1, a heme-Fe-containing protein) and NADPH cytochrome P450 reductase (RED, a FAD- and FMN-containing protein). The reaction consumes progesterone, molecular oxygen and NADPH.
For the conversion of 17xcex1-hydroxyprogesterone into 17xcex1,21-dihydroxy-xcex944-pregnene-3,20-dione (cortexolone), also two proteins are needed: steroid-21-hydroxylase (P450C21, a heme-Fe-containing protein) and the before-mentioned protein RED. The reaction consumes 17xcex1-hydroxy progesterone, molecular oxygen and NADPH.
In the conversion of cortexolone into hydrocortisone, three proteins are involved: steroid 11xcex2-hydroxylase (P45011xcex2, a heme-Fe-containing protein), and the above mentioned proteins ADX and ADR.
As described above, cytochrome P450 proteins are enzymes which are essential for the biochemical conversion of cholesterol to hydrocortisone. These enzymes belong to a larger group of cytochrome P450 proteins (or shortly P450 proteins). They have been encountered in prokaryotes (various bacteria) and eukaryotes (yeasts, molds, plants and animals). In mammals, high levels of P450 proteins are found in the adrenal cortex, ovary, testes and liver.
Many of these proteins, have been purified and are well characterized now. Their specific activity has been determined. Recently, a number of reviews on this subject have been published such as K. Ruckpaul and H. Rein (eds), xe2x80x9cCytochrome P450xe2x80x9d and P. R. Oritz de Montellano (ed.) xe2x80x9cCytochrome P450, structure, mechanism and biochemistryxe2x80x9d. Cytochrome P450 proteins are characterized by their specific absorbance maximum at 450 nm after reduction with carbon monoxide. In prokaryotic organisms, the P450 proteins are either membrane bound or cytoplasmatic. As far as the bacterial, P450 proteins have been studied in detail (e.g. P450meg and P450cam), it has been shown that a ferredoxin and a ferredoxin reductase are involved in the hydroxylating activity. For eukaryotic organisms, two types of P450 proteins, I and II have been described. Their two differences reside in:
1. subcellular localization, type I is localized in the microsomal fraction and type II is localized in the inner membrane of mitochondria;
2. the way the electrons are transferred to the P450 protein. Type I is reduced by NADPH via a P450 reductase, whereas Type II is reduced by NADPH via a ferredoxin-reductase (e.g. adrenodoxin reductase) and a ferredoxin (e.g. adrenodoxin).
According to EP-A-0,281,245, cytochrome P450 enzymes can be prepared from Streptomyces species and used for the hydroxylation of chemical compounds. The enzymes are used in isolated form, which is a rather tedious and expensive procedure.
JP-A-62,236,485 (Derwent 87-331,234) teaches that it is possible to introduce into Saccharomyces cerevisiae the genes of liver cytochrome P450 enzymes and to express them affording enzymes which may be used for their oxidation activity. However, in the above references, there is no indication of the use of cytochrome P450 enzymes for the preparation of steroid compounds.
It is an object of the invention to provide an improved biochemical pathway for the production of hydrocortisone and expression cassettes useful therein.
It is another object of the invention to provide recombinant host cells and their progeny containing said expression cassettes.
These and other objects and advantages of the invention will become obvious from the following detailed description.
The process of the invention for the preparation of hydrocortisone from sterols comprises culturing a recombinant cell in a nutrient medium, the recombinant host containing an expression cassette, operable in a recombinant host, comprising a heterologous DNA coding sequence encoding a protein, which is functional, alone or in cooperation with one or more additional proteins, of catalyzing an oxidation step in the biological pathway for conversion of cholesterol into hydrocortisone, which step is selected from the group consisting of:
the conversion of cholesterol to pregnenolone;
the conversion of pregnenolone to progesterone;
the conversion of progesterone to 17xcex1-hydroxy-progesterone;
the conversion of 17xcex1-hydroxyprogesterone to cortexolone;
the conversion of cortexolone to hydrocortisone, and the corresponding control sequences effective in said host.
The invention provides a multiplicity of expression cassettes for production of proteins necessary in, the construction of a multigenic system for the one-step conversion of inexpensive steroid starting materials to more rare and expensive end products, wherein such conversion is carried out in native systems through a multiplicity of enzyme-catalyzed and cofactor-mediated conversions, such as the production of hydrocortisone from cholesterol. The expression cassettes of the invention are useful in the ultimate production of multigenic systems for conducting these multi-step conversions.
Accordingly, in one aspect, the invention is directed to an expression cassette effective in a recombinant host cell in expressing a heterologous coding DNA sequence, wherein said coding sequence encodes an enzyme which is able, alone or in cooperation with additional proteins, to catalyze an oxidation step in the biological pathway for the conversion of cholesterol to hydrocortisone. The expression cassettes of the invention, therefore, include those sequences capable of producing, in a recombinant host, the following proteins: side-chain cleaving enzyme (P450SCC); adrenodoxin (ADX); adrenodoxin reductase (ADR); 3xcex2-hydroxy steroid dehydrogenase/isomerase (3xcex2-HSD); steroid 17xcex1-hydrexylase (P45017xcex1); NADPH cytochrome P450 reductase (RED); steroid-21-hydroxylase (P450C21); and steroid 11xcex2-hydroxylase (P45011xcex2).
In other aspects, the invention is directed to recombinant host cells transformed with these vectors or with the expression cassettes off the invention, to methods to produce the above enzymes and to use these enzymes for oxidation, to processes to use said host cells for specific oxidations in a culture broth and to pharmaceutical compositions containing compounds prepared by said processes.